An inkjet-printed polysaccharide matrix for on-chip sample preparation in point-of-care cell counting chambers.

On-chip sample preparation in self-contained microfluidic devices is a key element to realize simple, low-cost, yet reliable in vitro diagnostics that can be carried out at the point-of-care (POC) with minimal training requirements by unskilled users. To address this largely unmet POC medical need, we have developed an optimized polysaccharide matrix containing the reagents which substantially improves our fully printed POC CD4 counting chambers for the monitoring of HIV patients. The simply designed counting chambers allow for capillary-driven filling with unprocessed whole blood. We carefully tailored a gellan/trehalose matrix for deposition by inkjet printing, which preserves the viability of immunostains during a shelf life of at least 3 months and enables controlled antibody release for intense and homogeneous immunofluorescent cell staining throughout the complete 60 mm2 image area within 30 min. Excellent agreement between CD4 counts obtained from our fully printed CD4 counting chambers and the gold standard, flow cytometry, is demonstrated using samples both from healthy donors and HIV-infected patients.

All-printed cell counting chambers with on-chip sample preparation for point-of-care CD4 counting.

We demonstrate the fabrication of fully printed microfluidic CD4 counting chips with complete on-chip sample preparation and their applicability as a CD4 counting assay using samples from healthy donors and HIV-infected patients. CD4 counting in low-income and resource-limited point-of-care settings is only practical and affordable, if disposable tests can be fabricated at very low cost and all manual sample preparation is avoided, while operation as well as quantification is fully automated and independent of the skills of the operator. Here, we show the successful use of (inkjet) printing methods both to fabricate microfluidic cell counting chambers with controlled heights, and to deposit hydrogel layers with embedded fluorophore-labeled antibodies for on-chip sample preparation and reagent storage. The maturation process of gelatin after deposition prevents antibody wash-off during blood inflow very well, while temperature-controlled dissolution of the matrix ensures complete antibody release for immunostaining after the inflow has stopped. The prevention of antibody wash-off together with the subsequent complete antibody release guarantees a homogeneous fluorescence background, making rapid and accurate CD4 counting possible. We show the successful application of our fully printed CD4 counting chips on samples from healthy donors as well as from HIV-infected patients and find an excellent agreement between results from our method and from the gold standard, flow cytometry, in both cases.

Inability of monoclonal anti-light chain antibody to detect clonal B-cells in a patient with follicular lymphoma by multicolor flow cytometry.

BACKGROUND: Our recent publication "Inability of a monoclonal anti-light chain antibody to detect clonal plasma cells in a patient with multiple myeloma by multicolor flow cytometry," underlined the importance of choice of antibodies to detect cytoplasmic light chains. Our present study extends this finding for detection of surface immunoglobulin (SIg) light chains on clonal B-cells. METHODS: Multicolor flow cytometry was used for analyzing bone marrow (BM) from a patient with a CD10-positive follicular lymphoma for infiltrating clonal B-cells. RESULTS: In the BM aspirate, B cells could be identified expressing CD19, CD10, and high levels of CD20. No SIg light chain expression was found on this population of B cells employing monoclonal antibodies. Re-analysis using polyclonal antibodies against SIg light chains, revealed presence of lambda light chains on the CD10positive B-cells. CONCLUSIONS: These data illustrate when antibodies against SIg light chains are employed for B-cell clonality assessment, polyclonal antibodies are preferred over monoclonal antibodies.

A ZMYM2-FGFR1 8p11 myeloproliferative neoplasm with a novel nonsense RUNX1 mutation and tumor lysis upon imatinib treatment.

The 8p11 myeloproliferative neoplasm (8p11 MPN) is a rare disorder that is molecularly characterized by fusions of diverse partners to the tyrosine kinase receptor gene FGFR1. It can rapidly transform to acute myeloid leukemia. Here we report on a case with a t(8;13)(p11.2;q12.1) ZMYM2-FGFR1 fusion, with massive tumor lysis upon tyrosine kinase inhibition with imatinib. Upon reevaluation, we detected trisomy 21 in addition to the translocation. Sequencing revealed a nonsense c.958C →T RUNX1 mutation both at diagnosis and disease progression, resulting in a p.Arg320X carboxyl-terminal truncated RUNX1 protein. This is the first report on an 8p11 MPN with a trisomy 21 RUNX1 mutation.

Inability of a monoclonal anti-light chain antibody to detect clonal plasma cells in a patient with multiple myeloma by multicolor flow cytometry.

BACKGROUND: Multicolor flow cytometry (MFC) is increasingly important for the diagnosis and minimal residual disease (MRD) assessment of patients with plasma cells (PC) dyscrasias, like multiple myeloma. Recently published information shows that immunophenotype of myeloma PC can change over time and normal PC are heterogeneous in the expression of CD19 and CD56. This implies that for a sensitive, reliable detection of MRD clonality assessment by the detection of cytoplasmic kappa and lambda light chains is advisable. METHODS: Eight-color MFC was used to detect normal and myeloma PC by the expression of CD38 and CD138. Analysis of additional surface antigens like CD45, CD19, CD56, CD27, and the intracellular immunoglobulin light chain distribution were used to differentiate polyclonal from clonal PC. RESULTS: Absence of cytoplasmic light chains expression in a PC subpopulation with an abnormal phenotype suggested the presence of non-secretory plasma cells in the bone marrow (BM) of this patient. This observation however, was contradicted by the presence of free lambda light chains in the patient's serum. After repeating the analysis with polyclonal antibodies against intracellular immunoglobulin light chains instead of monoclonal antibodies, the abnormal PC subpopulation appeared to express lambda light chains. CONCLUSION: These data illustrate that if clonality assessment of PC is included in disease monitoring, the use of polyclonal over monoclonal antibodies is preferred for the detection of intracellular immunoglobulin light chains.